A semi-defined medium has been developed for axenic cultivation of a strain of Entamoeba histolytica. The only undefined ingredient is a 0.5% of casein peptone digest. The serum component of TYI-S-33, a medium currently used worldwide for axenic cultivation of Entamoeba, has been replaced with a lipoprotein-cholesterol fraction of bovine serum and a commercially produced chemically defined serum substitute. Yields of amebae are equivalent to those obtained with whole serum. Conversion of an avirulent stain of E. histolytica characterized by a non-pathogenic zymodeme and isolated from an asymptomatic carrier case to a virulent strain was accomplished for the first time by suppressing the growth of the intestinal flora associated with the amebae and transferring the amebae to a medium for axenic cultivation. Conversely, the virulence of a highly virulent axenized strain of E. histolytica was suppressed when the ameba was associated with the bacterial flora derived from the avirulent strain. Reaxenization was followed by restoration of virulence. New evidence that E. histolytica and E. histolytica-like Laredo type amebae are not conspecific has been provided by comparing the ribosomal cistrons of the two organisms probed with the ribosomal cistron of Plasmodium lophurae.